Erythropoietin (EPO) is a glycoprotein hormone which stimulates red blood cells by a process known as erythropoiesis. EPO is produced in the kidney and stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. In patients with renal insufficiency, serum EPO levels remain low, inappropriately low serum EPO levels may also be seen in anemic patients with cancer, Human Immunodeficiency Virus (HIV) infection, ulcerative colitis and sickle cell anemia. For all these indications and to decrease the rate of blood transfusion, EPO is established as an effective treatment.
The rHu EPO is a 165 amino acid containing glycoprotein produced through recombinant DNA technology in animal cell lines such as Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK) cell lines. The recombinant human erythropoietin (rHu EPO) has the same biological properties as endogenous erythropoietin secreted in humans. It has a molecular weight of about 36,000 daltons with carbohydrate moiety composing about 30% of molecular weight.
Pegylation technology has emerged as a means to improve the pharmacokinetic and pharmcodynamic properties of biopharmaceuticals. Some of the benefits of pegylation include improved clinical properties, enhanced solubility, sustained absorption and release, reduced immunogenicity and proteolysis, reduced clearance from circulation by the kidneys, increased dosing intervals due to higher in-vivo half-lives owing to increased circulation time and the like. The longer circulation of Erythropoietin results in beneficial therapeutic effects such as prolongation of its presence in the human body, effective therapeutic treatment of disease and conditions thereof.
Pegylated Erythropoietin (MIRCERA® from Roche) is a Pegylated recombinant form of human EPO. The erythropoietin used to generate MIRCERA® is the active substance of Neorecormon® (epoetin beta; Roche's recombinant EPO first approved for general medical use in the EU in 1996). The PEG moiety used is methoxypolyethylene glycol-succinimidyl butanoic acid (PEG-SBA); a 30 kDa linear chemically activated PEG. The PEG-SBA spontaneously forms amide linkages with either EPO's N-terminal
amino group or with the E-amino group of an accessible surface lysine residue (Lys 45 or Lys 52). The final product generated is a 60 kDa monopegylated product.
U.S. Pat. No. 4,992,419 disclosed a compatible, storage-stable human protein preparation containing a human protein, a physiologically compatible buffer and optionally complex formers, isotonicity-adjusting agents, calcium chloride and other materials usual for injection purposes which, in an injectable form, contain 5 to 50 gm/liter urea, 1 to 50 gm/liter amino acid and 0.05 to 5 gm/liter non-ionic wetting agent. A process for the production of this preparation is also disclosed.
U.S. Pat. Nos. 6,120,761 and 7,011,825 disclosed an erythropoietin solution preparation containing an amino acid as a stabilizer, and having excellent long-term storage stability.
U.S. Pat. No. 7,202,208 disclosed a liquid pharmaceutical composition consisting essentially of an erythropoietin protein, a multiple charged inorganic anion in a pharmaceutically acceptable buffer suitable to keep the solution pH in the range from about 5.5 to about 7.0, and optionally one or more pharmaceutically acceptable excipients. This composition is especially useful for the prophylaxis and treatment of diseases related to erythropoiesis.
U.S. Pat. No. 7,842,661 disclosed conjugates between erythropoietin and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.
IN220067 disclosed a new stable pharmaceutical composition of erythropoietin (EPO) that is stabilized with a combination of a poloxamer polyol and a polyhydric alcohol.
IN234438 disclosed an aqueous formulation of human erythropoietin, comprising the human erythropoietin of the kind such as herein described and the range of 100 IU/ml to 120,000 IU/ml; non-ionic surfactant of the kind such as herein described and the range of 0.0001 to 0.01% (w/v), polyhydric alcohol of the kind such as herein described and the range of 0.001 to 2% (w/v), neutral amino acid of the kind such as herein described and the range of 0.001 to 2% (w/v) and sugar alcohol of the kind such as herein described and the range of 0.1 to 1.0% (w/v) as stabilizers; isotonic reagent of the kind such as herein described and the range of 0.001 to 0.7% (w/v); and buffering reagent of the kind such as herein described and the range of 1 mM to 50 mM and the range of pH 6.0 to 7.5.
However, the bioavailability of commercially available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining maximum clinical potency.
Despite the revolutionary progress in the large-scale manufacturing of proteins for therapeutic use, effective and convenient delivery of these agents in the body remains a major challenge due to their intrinsic physicochemical properties such as large molecular size, self association, physical and chemical instability, aggregation and adsorption. Hence, a novel formulation is required which can overcome all such instability of the formulation available in prior art.